Influence of cholesterol and ergosterol on membrane dynamics using different fluorescent reporter probes

Ergosterol is an evolutionary precursor of cholesterol and is the major sterol present in lower eukaryotes. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is still at a very early stage. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing fluorescent reporter probes pyrene and TMA-DPH. These results show, for the first time, the important differences on the effect of cholesterol and ergosterol in short-range ordering (reported by TMA-DPH) and long-range dynamics (reported by pyrene). In addition, pyrene vibronic peak intensity ratio provides information on polarity of the microenvironment experienced by the probe. These novel results are relevant in the context of membrane domains in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Amphidinol 3 preferentially binds to cholesterol in disordered domains and disrupts membrane phase separation

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 μM, as in the case of methyl-β-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.     

Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol

Sterol molecules are essential for maintaining the proper structure and function of eukaryotic cell membranes. The influence of cholesterol (the principal sterol of higher animals) on the lipid bilayer properties was extensively studied by both experimental and simulation methods. In contrast, the effect of ergosterol (the principal fungal sterol) on the membrane structure and dynamics is much less recognized. This work presents the results of comparative molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine bilayer containing approximately 25 mol % of cholesterol or ergosterol. A detailed analysis of the molecular properties (e.g., bilayer thickness, lipid order, diffusion, intermolecular interactions, etc.) of both sterol-induced liquid-ordered membrane phases is presented. Presence of sterols in the membrane significantly changes its property, especially fluidity and molecular packing. Moreover, in accordance with the experiments, our calculations show that, compared to cholesterol, ergosterol has higher ordering effect on the phospholipid acyl chains. This different influence on the properties of the lipid bilayer stems from differences in conformational freedom of sterol side chains. Additionally, obtained models of lipid membranes containing human and fungal sterols, constituting the result of our work, can be also utilized in other chemotherapeutic studies on interaction of selected ligands (e.g., antifungal compounds) with membranes.     

Zwitterionic phospholipids and sterols modulate antimicrobial peptide-induced membrane destabilization

Cationic amphipathic α-helical peptides preferentially disrupt anionic lipids in mixed model membranes, potentially causing a catastrophic release of the cell contents or attenuation of the membrane potential. The effective role of such peptides requires considerable discrimination between target and host cells, which is likely to occur at the level of the cell membrane. Here, we explore the roles of a variety of common membrane constituents in mediating the interaction between the antimicrobial peptide pleurocidin and model membranes. We employ intrinsic tryptophan fluorescence and circular dichroism to observe the effect of increasing concentrations of sterol in the membrane on peptide binding, using ²H solid-state NMR of chain deuterated lipids simultaneously to probe the effective chain disruption of the anionic phospholipid component of the membrane. We show that the degree of ordering of the lipid acyl chains in the membrane is dependent on the nature of the zwitterionic phospholipid headgroup in mixed anionic membranes. Furthermore, the presence of cholesterol and ergosterol increases acyl chain order in the liquid crystalline model membranes, but to differing degrees. Our results show how sterols can protect even negatively charged membranes from the disruptive effects of antimicrobial peptides, thereby providing a molecular view of the differences in sensitivity of various target membranes to linear cationic antibiotic peptides where bacteria (no sterols) are most susceptible, lower eukaryotes including fungi (containing ergosterol) exhibit an intermediate degree of sensitivity, and higher organisms (containing cholesterol) are largely resistant to antimicrobial peptides.     

Sterol carrier protein-2 selectively alters lipid composition and cholesterol dynamics of caveolae/lipid raft vs nonraft domains in L-cell fibroblast plasma membranes

Although the functional significance of caveolae/lipid rafts in cellular signaling and cholesterol transfer is increasingly recognized, almost nothing is known regarding the lipids, cholesterol dynamics, and factors regulating these properties in caveolae/lipid rafts as opposed to nonlipid raft domains of the plasma membrane. The present findings demonstrate the utility of con-A affinity chromatography for simultaneous isolation of caveolae/lipid raft and nonlipid raft domains from plasma membranes of L-cell fibroblasts. These domains differed markedly in both protein and lipid constituents. Although caveolae/lipid rafts were enriched in total lipid, cholesterol, and phospholipid as well as other markers for these domains, the cholesterol/phospholipid ratio of caveolae/lipid rafts did not differ from that of nonlipid rafts. Nevertheless, spontaneous sterol transfer was 7-12-fold faster from caveolae/lipid raft than nonlipid raft domains of the plasma membrane. This was largely due to the near absence of exchangeable sterol in the nonlipid rafts. SCP-2 dramatically and selectively enhanced sterol transfer from caveolae/lipid rafts, but not from nonlipid rafts. Finally, overexpression of SCP-2 significantly altered the sterol dynamics of caveolae/lipid rafts to facilitate retention of cholesterol within the cell. These results established for the first time that (i) caveolae/lipid rafts, rather than the nonlipid raft domains, contain significant levels of rapidly transferable sterol, consistent with their role in spontaneous sterol transfer from and through the plasma membrane, and (ii) SCP-2 selectively regulates how caveolae/lipid rafts, but not nonlipid raft domains, mediate cholesterol trafficking through the plasma membrane.     

Long Open Amphotericin Channels Revealed in Cholesterol-Containing Phospholipid Membranes Are Blocked by Thiazole Derivative

The action of antifungal drug, amphotericin B (AmB), on solvent-containing planar lipid bilayers made of sterols (cholesterol, ergosterol) and synthetic C14–C18 tail phospholipids (PCs) or egg PC has been investigated in a voltage-clamp mode. Within the range of PCs tested, a similar increase was achieved in the lifetime of one-sided AmB channels in cholesterol- and ergosterol-containing membranes with the C16 tail PC, DPhPC at sterol/DPhPC molar ratio ≤1. The AmB channel lifetimes decreased only at sterol/DPhPC molar ratio >1 that occurred with sterol/PC molar ratio of target cell membranes at a pathological state. These data obtained on bilayer membranes two times thicker than one-sided AmB channel length are consistent with the accepted AmB pore-forming mechanism, which is associated with membrane thinning around AmB–sterol complex in the lipid rafts. Our results show that AmB can create cytotoxic (long open) channels in cholesterol membrane with C14–C16 tail PCs and nontoxic (short open) channels with C17–C18 tail PCs as the lifetime of one-sided AmB channel depends on ~2–5 Å difference in the thickness of sterol-containing C16 and C18 tail PC membranes. The reduction in toxic AmB channels efficacy can be required at the drug administration because C16 tails in native membrane PCs occur almost as often as C18 tails. The comparative analysis of AmB channel blocking by tetraethylammonium chloride, tetramethylammonium chloride and thiazole derivative of vitamin B₁, 3-decyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl) thiazole chloride (DMHT), has proved that DMHT is a comparable substitute for both tetraalkylammonia that exhibits a much higher affinity.     

Differential effect of cholesterol and its biosynthetic precursors on membrane dipole potential

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, a representative sterol in higher eukaryotic membranes, is known to increase membrane dipole potential. In this work, we explored the effects of immediate (7-DHC and desmosterol) and evolutionary (ergosterol) precursors of cholesterol on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that the effect of these precursors on membrane dipole potential is very different from that observed with cholesterol, although the structural differences among them are subtle. These results assume relevance, since accumulation of cholesterol precursors due to defective cholesterol biosynthesis has been reported to result in several inherited metabolic disorders such as the Smith-Lemli-Opitz syndrome. Interestingly, cholesterol (and its precursors) has a negligible effect on dipole potential in polyunsaturated membranes. We interpret these results in terms of noncanonical orientation of cholesterol in these membranes. Our results constitute the first report on the effect of biosynthetic and evolutionary precursors of cholesterol on dipole potential, and imply that a subtle change in sterol structure can significantly alter the dipolar field at the membrane interface.     

How cholesterol interacts with proteins and lipids during its intracellular transport

Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein–lipid and protein–protein interactions. Similarly, membrane lipids and their physico-chemical properties directly affect cholesterol partitioning and thereby contribute to the highly heterogeneous intracellular cholesterol distribution. Movement of cholesterol in cells is mediated by vesicle trafficking along the endocytic and secretory pathways as well as by non-vesicular sterol exchange between organelles. In this article, we will review recent progress in elucidating sterol–lipid and sterol–protein interactions contributing to proper sterol transport in living cells. We outline recent biophysical models of cholesterol distribution and dynamics in membranes and explain how such models are related to sterol flux between organelles. An overview of various sterol-transfer proteins is given, and the physico-chemical principles of their function in non-vesicular sterol transport are explained. We also discuss selected experimental approaches for characterization of sterol–protein interactions and for monitoring intracellular sterol transport. Finally, we review recent work on the molecular mechanisms underlying lipoprotein-mediated cholesterol import into mammalian cells and describe the process of cellular cholesterol efflux. Overall, we emphasize how specific protein–lipid and protein–protein interactions help overcoming the extremely low water solubility of cholesterol, thereby controlling intracellular cholesterol movement. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Fluorescence and multiphoton imaging resolve unique structural forms of sterol in membranes of living cells

Although cholesterol is an essential component of mammalian membranes, resolution of cholesterol organization in membranes and organelles (i.e. lysosomes) of living cells is hampered by the paucity of nondestructive, nonperturbing methods providing real time structural information. Advantage was taken of the fact that the emission maxima of a naturally occurring fluorescent sterol (dehydroergosterol) were resolvable into two structural forms, monomeric (356 and 375 nm) and crystalline (403 and 426 nm). Model membranes (sterol:phospholipid ratios in the physiological range, e.g. 0.5-1.0), subcellular membrane fractions (plasma membranes, lysosomal membranes, microsomes, and mitochondrial membranes), and lipid rafts/caveolae (plasma membrane cholesterol-rich microdomain purified by a nondetergent method) contained primarily monomeric sterol and only small quantities (i.e. 1-5%) of the crystalline form. In contrast, the majority of sterol in isolated lysosomes was crystalline. However, addition of sterol carrier protein-2 in vitro significantly reduced the proportion of crystalline dehydroergosterol in the isolated lysosomes. Multiphoton laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured with dehydroergosterol for the first time provided real time images showing the presence of monomeric sterol in plasma membranes, as well as other intracellular membrane structures of living cells. Furthermore, MPLSM confirmed that crystalline sterol colocalized in highest amounts with LysoTracker Green, a lysosomal marker dye. Although crystalline sterol was also detected in the cytoplasm, the extralysosomal crystalline sterol did not colocalize with BODIPY FL C(5)-ceramide, a Golgi marker, and crystals were not associated with the cell surface membrane. These noninvasive, nonperturbing methods demonstrated for the first time that multiple structural forms of sterol normally occurred within membranes, membrane microdomains (lipid rafts/caveolae), and intracellular organelles of living cells, both in vitro and visualized in real time by MPLSM.     

Universal behavior of membranes with sterols

Lanosterol is the biosynthetic precursor of cholesterol and ergosterol, sterols that predominate in the membranes of mammals and lower eukaryotes, respectively. These three sterols are structurally quite similar, yet their relative effects on membranes have been shown to differ. Here we study the effects of cholesterol, lanosterol, and ergosterol on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid bilayers at room temperature. Micropipette aspiration is used to determine membrane material properties (area compressibility modulus), and information about lipid chain order (first moments) is obtained from deuterium nuclear magnetic resonance. We compare these results, along with data for membrane-bending rigidity, to explore the relationship between membrane hydrophobic thickness and elastic properties. Together, such diverse approaches demonstrate that membrane properties are affected to different degrees by these structurally distinct sterols, yet nonetheless exhibit universal behavior.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

Selective cholesterol dynamics between lipoproteins and caveolae/lipid rafts

Although low-density lipoprotein (LDL) receptor-mediated cholesterol uptake through clathrin-coated pits is now well understood, the molecular details and organizing principles for selective cholesterol uptake/efflux (reverse cholesterol transport, RCT) from peripheral cells remain to be resolved. It is not yet completely clear whether RCT between serum lipoproteins and the plasma membrane occurs primarily through lipid rafts/caveolae or from non-raft domains. To begin to address these issues, lipid raft/caveolae-, caveolae-, and non-raft-enriched fractions were resolved from purified plasma membranes isolated from L-cell fibroblasts and MDCK cells by detergent-free affinity chromatography and compared with detergent-resistant membranes isolated from the same cells. Fluorescent sterol exchange assays between lipoproteins (VLDL, LDL, HDL, apoA1) and these enriched domains provided new insights into supporting the role of lipid rafts/caveolae and caveolae in plasma membrane/lipoprotein cholesterol dynamics: (i) lipids known to be translocated through caveolae were detected (cholesteryl ester, triacylglycerol) and/or enriched (cholesterol, phospholipid) in lipid raft/caveolae fractions; (ii) lipoprotein-mediated sterol uptake/efflux from lipid rafts/caveolae and caveolae was rapid and lipoprotein specific, whereas that from non-rafts was very slow and independent of lipoprotein class; and (iii) the rate and lipoprotein specificity of sterol efflux from lipid rafts/caveolae or caveolae to lipoprotein acceptors in vitro was slower and differed in specificity from that in intact cells-consistent with intracellular factors contributing significantly to cholesterol dynamics between the plasma membrane and lipoproteins.     

Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.     

Deuterium NMR Study of the Effect of Ergosterol on POPE Membranes

We study the effect of ergosterol on the physical properties of 1-[²H₃₁]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multibilayers using deuterium nuclear magnetic resonance. NMR spectra were taken as a function of temperature and ergosterol concentration up to 70 mol %. The spectral first moments show that there is a dramatic difference in the ability of ergosterol to disorder the gel phase and to order the liquid-crystalline phase of POPE membranes, an unusual behavior among lipid/sterol systems studied up to now. Further investigation of the liquid-crystalline phase shows that ergosterol (erg) increases the chain order of POPE-d31, but that this effect saturates at 10 mol % ergosterol. This is in marked contrast to the effect of cholesterol (chol) on POPE membranes: the chain order of POPE increases with cholesterol to at least 45 mol %. Moreover, we found that at higher ergosterol concentrations (>40 mol %) ergosterol decreases the POPE-d31 chain order, which, to our knowledge, has not been directly observed in other lipid/sterol systems. The temperature-composition phase diagram is presented. Finally, at all ergosterol concentrations, the chain order of liquid-crystalline-phase POPE is much smaller than that of comparable POPE/chol membranes. This implies that there is no liquid-ordered phase behavior for POPE/erg membranes.     

Fluorescent sterols as tools in membrane biophysics and cell biology

Cholesterol is an important constituent of cellular membranes playing a fundamental role in many biological processes. This sterol affects membrane permeability, lateral lipid organization, signal transduction and membrane trafficking. Intracellular sterol transport modes and pathways as well as the regulation of sterol metabolism and disposition in various tissues are areas of intense research. Progress is intimately linked to development and use of appropriate analogs, which closely mimic the properties of cholesterol while allowing to be detected by spectroscopic or microscopic methods. This review provides an overview of various fluorescent sterols used in membrane biophysics and cell biology including analogs of cholesterol and cholesteryl esters. Attention is paid to the natural fluorescent sterol dehydroergosterol (DHE). A survey of the many applications of DHE in biological research is presented. Special emphasis is on recent developments in fluorescence microscopy instrumentation to visualize DHE as an intrinsically fluorescent analog of cholesterol in living cells.     

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.     

Mechanisms of sterol uptake and transport in yeast

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. Article from a special issue on Steroids and Microorganisms.     

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement

Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells. In our previous study, the effect of methyl-beta-cyclodextrin (MbCD), cholesterol-sequestering agent, on Ca²⁺-permeable stretch-activated cation channels (SACs) has been described. Here, cell-attached patch-clamp method was employed to search for the mechanisms of cholesterol-dependent regulation of SACs and to clarify functional contribution of lipid bilayer and submembranous cytoskeleton to channel gating. Cholesterol-depleting treatment with MbCD significantly decreased open probability of SACs whereas alpha-cyclodextrin had no effect. F-actin disassembly fully restored high level of SAC activity in cholesterol-depleted cells. Particularly, treatment with cytochalasin D or latrunculin B abrogated inhibitory effect of MbCD on stretch-activated currents. Single channel analysis and fluorescent imaging methods indicate that inhibition of SACs after cholesterol depletion is mediated via actin remodeling initiated by disruption of lipid rafts. Our data reveal a novel mechanism of channel regulation by membrane cholesterol and lipid rafts.     

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L

Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X(Sterol)=0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol>lanosterol> or =ergosterol, and cholesterol>lanosterol>ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.